AN OBLIQUE-INCIDENCE REFLECTIVITY DIFFERENCE STUDY OF THE DEPENDENCE OF PROBE-TARGET REACTION CONSTANTS ON SURFACE TARGET DENSITY USING STREPTAVIDIN-BIOTIN REACTIONS AS A MODEL

Abstract

A combination of the microarray platform and oblique-incidence reflectivity difference (OI-RD) microscopy was used to study the dependence of kinetic constants of probe-target reactions on the surface density of immobilized targets. Streptavidin-biotin reactions with a very high binding affinity were employed as the study model. Oblique-incidence reflectivity difference microscopy, a label-free and surface-based detection technique, was developed for monitoring real-time binding curves between two interactive biomolecules, enabling the acquisition of kinetic constants such as on-rate, off-rate, and equilibrium dissociation constants. These kinetic constants are important in characterizing biomolecular interactions because in living cells all intercellular and intermolecular reactions are at dynamic rather than at stable equilibrium. The kinetic constant of streptavidin binding to surface-immobilized biotin-bovine serum albumin was demonstrated to be significantly affected by the density of surface bovine serum albumin conjugates, mainly due to mass-transport effects within targets.